XPF-ERCC1 Participates in the Fanconi Anemia Pathway of Cross-Link Repair
XPF-ERCC1 Participates in the Fanconi Anemia Pathway of Cross-Link Repair
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DOI：
10.1128/mcb.00086-09
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发表时间：
2009-12-15
影响因子：
5.3
通讯作者：
McHugh, Peter J.
中科院分区：
文献类型：
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作者：
Bhagwat, Nikhil;Olsen, Anna L.;McHugh, Peter J.
链间交联 (ICL) 可防止 DNA 链分离，从而阻止转录和复制，使其具有极强的细胞毒性。 ICL 从哺乳动物基因组中去除的精确机制在很大程度上仍然难以捉摸。遗传证据表明 ATR、范可尼贫血蛋白、同源重组、跨损伤合成所需的蛋白以及至少两种核酸内切酶 MUS81-EME1 和 XPF-ERCC1。 ICL 会导致复制依赖性 DNA 双链断裂 (DSB)，而 MUS81-EME1 会促进 DSB 形成。 ICL 被 XPF-ERCC1 脱钩后，这些 DSB 的后续修复通过同源重组发生。在这里，我们检查了任一核酸酶的丢失对 FANCD2 单泛素化的影响，以确定 ICL 的溶核过程是否是激活范可尼贫血途径所必需的。 FANCD2 在暴露于交联剂的 Mus81(-/-)、Ercc1(-/-) 和 XPF 缺陷的人类、小鼠和仓鼠细胞中单泛素化。然而，FANCD2 的单泛素化形式在 XPF-ERCC1 缺陷细胞中比在野生型细胞中持续时间更长。此外，在 XPF-ERCC1 缺陷细胞中，染色质结合的 FANCD2 水平显着降低，并且 ICL 诱导的 FANCD2 灶的数量显着降低。这些数据表明，XPF-ERCC1 对 ICL 的脱钩对于 FANCD2 稳定定位到染色质以及随后的同源重组介导的 DSB 修复是必要的。
Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81(-/-), Ercc1(-/-), and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.